DNase I digestion of DNA
Bovine pancreatic deoxyribonuclease I (DNase I) attacks the DNA phosphate backbone from the minor groove, with a cleavage rate that decreases where the minor groove is narrow (12
). It has been widely used to probe detailed sequence-dependent structural variations (14
). Three 68-mer double-stranded DNAs containing the E2-binding site with different spacer sequences, ATAT, TATA and ACGT, were used in the present study. The 5′-33
P-end labelled DNA was digested by DNase I and the hydrolysis products were analysed by electrophoresis on sequencing gels.
An autoradiograph of the patterns of digestion of the different E2 target sequences in the presence of Mg2+ is shown in a. It can be seen that DNase I digestion produces essentially the same gel patterns except in regions containing the different binding sites. The intensities of the cleavage products in Mg2+, measured by scanning densitometry in the binding-site domains, are shown in . All three spacer variants display very similar cutting patterns in the 5′-ACCG sequence, which lies 5′ to the spacer sequence. As expected, the spacer sequences show distinctive patterns, with relatively strong cleavage at ApT steps in the ATAT and TATA spacers.
DNase I footprinting of the 5′-end labelled complementary strand containing different E2-binding spacer (ATAT, TATA and ACGT) in presence of (a) Mg2+ and (b) Ca2+. Spacer regions are indicated in boxed area.
Ratio of intensity and total intensities (%) of DNase I cleavage measured by scanning densitometry in the regions of different protein-binding sites (a) ATAT, (b) TATA and (c) ACGT in the presence of 10 mM Mg2+.
In contrast, cutting at the CpG dinucleotide is highly variable. As shown in , weak cutting is observed in the 5′-half of the binding site, where the sequences are (C)CpG(A), (C)CpG(T) and (C)CpG(A) in the ATAT, TATA and ACGT spacers, respectively. On the other hand, cutting is strong at CpG in the complementary sequence in the 3′-portion of the binding site for the ATAT and ACGT spacers, for which both sequences are (T)CpG(G). The outlier is the TATA spacer, with sequence (A)CpG(G), for which cutting is weak. A similar pattern of DNase I cutting in the central 8 bp of the ATAT and TATA spacer sequences was observed by Fox (16
Thus DNase I cleavage at the phosphate group in the CpG 3′ binding-site sequence is strongly affected by the neighbouring spacer, reflecting a local alteration of DNA backbone geometry transmitted from spacer nucleotides 5′ to the CpG dinucleotide. Reduction in the cleavage rate at CpG presumably reflects a narrowing of the minor groove in the binding site. It is reasonable to infer that this structural effect perturbs the geometry of the major groove, to which an α-helix from the E2 protein is bound in the complex (17
), thereby reducing the binding affinity. Since the binding site is 2-fold symmetric, the CGGT sequence on the other strand should be similarly affected. On the other hand, DNase I cleavage does not reveal any significant spacer-dependent structural difference in the ACCG strand in the 5′-half of the binding site. It is notable that the C in the CpG step 3′ to the spacer is variable in the E2-binding sites on the papilloma virus genomes (18
The weakly cut CpG in the binding site of the TATA spacer molecule is embedded in an ACG trinucleotide, whereas strong cutting is displayed at the TCG trinucleotide in the other two spacer sequences. A similar effect of weak cutting is also displayed by the ACG trinucleotide of the central ACGT spacer. It is unlikely, however, that the structural effect can be explained entirely on the basis of the ACG trinucleotide, since the spacer sequence TTAA, which contributes this trinucleotide to the adjacent CpG, showed binding affinity within a factor 2 of the value expected on the basis of its curvature and mechanical properties (10
The effect of Ca2+ on the patterns of cutting by DNase I was also investigated. band reveal clear similarities in cutting pattern to that observed in the presence of Mg2+, including reduced cutting at CpG adjacent to the TATA spacer.
Ratio of intensity and total intensities (%) of DNase I cleavage measured by scanning densitometry in the regions of different protein-binding sites (a) ATAT, (b) TATA and (c) ACGT in the presence of 10 mM Ca2+.
Sequence-dependent Mg2+-induced curvature
Considerable interest has focused on sequence-specific and ion-dependent divalent cation association with DNA molecules (19–22
), which is thought to affect DNA bending and flexibility. Therefore, the interplay between DNA global structure and mechanical properties, cation binding and E2 protein association is intriguing, but has not been specifically investigated in solution.
We compared apparent DNA bending in the absence and presence of Mg2+
utilizing DNA gel migration-anomaly assay for a series of DNA ladders constituted by the E2-binding sites ligated approximately in phase with their helical repeats, with results shown in . The presence of Mg2+
retards DNA migration, reflecting reduced net charge. This effect can be removed by normalizing the mobilities to a standard sequence, as shown in . The extent of the relative retardation varies amongst DNA sequences, mainly depending on apparent curvature; TATA exhibits the largest shift when Mg2+
is added, indicating the largest static and/or dynamical structural sensitivity to Mg2+
, whereas ACGT shows the smallest. However, the induced apparent curvature is small when compared with the known (weak) DNA-bending element AATT () as also shown by the crystal structure of the dodecameric E2 DNA target, ACCGAATTCGGT (23
Figure 4. Magnesium enhances DNA curvature. Comparisons of DNA migration anomaly in the absence and presence of magnesium. The gel mobility retardation (R) is expressed as ratio of the migration distance of a control DNA ladder to that of indicated DNA molecule. (more ...)
Sequence-dependent ion release upon E2 binding
In seeking to understand the weaker binding of E2 to DNA containing the TATA spacer in Mg2+
than predicted on the basis of the curvature and flexibility of the spacer DNA (10
), we have investigated the thermodynamic linkage between binding of E2 and a series of ions (K+
) by measuring protein-binding affinity as a function of ion concentration. In its simplest form, in which only cation release is considered, the stoichiometric equation
describing the release of n
cations M of charge j+
leads to the theoretical dependence of dissociation constant Kd
on ion concentration [M] according to
It should be noted that the released ions upon protein binding may come not only from DNA, but also from protein; comparative values from one spacer sequence to another are more significant than absolute numbers.
shows the primary data, and summarizes the slopes of the lines in , giving the apparent number of ions released. The TATA spacer is clearly exceptional in that the maximal number of Mg2+
is displaced, by a margin that well exceeds experimental errors. By contrast, in the presence of Ca2+
, there is no clear difference in n
between TATA and ATAT spacers. Except for TATA, GC-rich spacer sequences tend to release more divalent cations than AT-rich sequences, consistent with the general observation of Chiu and Dickerson (22
) that Ca2+
show a preference for binding GC-rich sequence elements compared to AT. Chiu and Dickerson also found that Ca2+
have different modes of binding to localized sequence elements in identical crystal packing environments, which provides a plausible basis for the differential release of Ca2+
from identical spacer sequences.
Figure 5. Variation of binding constant with (a) Mg2+, (b) Na+, (c) Ca2+ or (d) K+ concentration. The number of cation release can be calculated by n = logKd/log[cation], or slope of the fitted straight line as shown, with results exhibited in (more ...)
Number of Mg2+, Ca2+, Na+ or K+ released upon DNA-E2 binding
Divalent cations can better hydrate well-ordered water molecules than can monovalent cations, leading to their sequence-specific hydrogen bonding with DNA bases and phosphates (20–22
), in addition to their direct electrostatic interactions. To test this valence-specific effect, divalent cations in the binding buffer were replaced with monovalent cations (Na+
) and their n
values were determined. As shown in , ion release varied only slightly over the sequences tested, implying that the possible sequence-dependent variation of the electrical potential of the DNA surface does not result in variations of monovalent cation binding.
and , which report the ratio of Kd
values for the closely related TATA and ATAT spacer sequences for the ion series studied, provide further insight into the underlying ion-dependent phenomena. It is notable that the Kd
ratio increases strongly in the order of increasing positive charge density on the ions, in the order K+
. In K+
, the relative affinity is within experimental error of the value (~2) predicted on the basis of the relative curvature and flexibility values (measured in the presence of Mg2+
). For the ions K+
, there is no significant systematic dependence of relative binding constant on ion concentration. We conclude that K+
interfere increasingly with binding to TATA over ATAT, but that this effect is not due to an increase in direct competition for binding sites in one sequence over the other. On the other hand, in Mg2+
ratio increases ~30-fold for a ~2-fold increase in ion concentration, implying that 5–6 more Mg2+
ions compete with E2 for binding to the TATA sequence compared to ATAT.
Ratio of dissociation constants for TATA and ATAT spacer constructs
Ratio of TATA and ATAT dissociation constants in Mg2+
Thus, there appear to be two components to the phenomenon at hand. First, ions of increasing positive charge density increasingly distort DNA structure in a way that weakens protein affinity, but protein binding either (or both) does not require dissociation of the K+, Na+ and Ca2+ ions that induce this effect, or results in equal displacement of these ions from the two sequences. The former could be the case, for example, if the ions interact with the minor groove, while the protein occupies the major groove. Second, in Mg2+ solution, 5–6 additional ions bind to TATA compared to ATAT in a way that directly competes with binding. This could result, for example, from additional ion binding to the major groove at positions contacted by the protein.
In structural terms, the anomaly shown by the TATA spacer may be related to previous observations that this particular tetranucleotide can adopt an A-DNA-like structure characterized by a wide and shallow minor groove either alone, or when bound to the TATA-box-binding protein (24
). The structure of the TATA-containing octamer (GGTATACC), in its complex with DNase I, also exhibited A-like features characterized by a wide and shallow minor groove (12
) whereas other sequences bound to the enzyme exhibited the B-type conformation (25
). Such conformational variations may be related to the different cutting pattern of the TATA spacer in comparison to that of the ATAT and ACGT spacer sequences. The ApC step in the DNase I complex of GGTATACC is the (putative) cutting site (12
), analogous to the strong cutting site ApC in the TATA spacer construct. The observation that this site was not cleaved in the complex of the crystal structure could be related to the excess of EDTA and other ingredients used for crystallization (12
). We note that the very strong cutting sites in the three constructs seem always to be flanked by quite weak sites, which raises the possibility that there may be local competition effects for binding/cutting. In this interpretation, the weak cutting at CpG would be a consequence of competition from the strong cutting at the adjacent ApC, supporting the view that the phenomenon results primarily from the shift to A-like geometry induced by the TATA sequence.
The transition from the TATA element structure with A-type characteristics, wide and shallow minor groove, to the one required to form the complex with the E2 protein, namely, a narrow and deep minor groove caused by DNA bending towards the protein, could account for the relatively lower binding affinity of this sequence. The presence of cations may further distort the DNA helix, thus explaining the observed increase in the Kd
and the large release of Mg2+
ions upon E2 binding in comparison to the other sequences. Whereas it is well known that Mg2+
ions are crucial for RNA folding, their roles in mediating DNA structural variation are generally neglected. Our work corroborated results from previous studies and suggested active roles of Mg2+
ions in sequence-specific DNA structures and protein–DNA interactions (26